C-terminal fragment of tetanus toxin (Hc) for treatment of depression

ABSTRACT

C-terminal domain of the heavy chain of tetanus toxin (Hc-TeTx) provides therapeutic effects in motor impairments associated with Parkinson disease (PD), and provides long lasting antidepressant effects, thus useful in treating and mitigating depression, particularly PD-depression co-morbid condition. A method for treating or mitigating depression, including administrating an effective amount of C-terminal domain of the heavy chain of tetanus toxin (Hc-TeTx) to a subject in need thereof. A method for treating or mitigating motor impairments associated with Parkinson&#39;s disease (PD), including administrating an effective amount of C-terminal domain of the heavy chain of tetanus toxin (Hc-TeTx) to a subject in need thereof.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Some aspects of this invention were made in the course of GrantNIH/NIAAA R03AA022479 awarded by the National Institutes of Health andtherefore the Government has certain rights in some aspects of thisinvention.

TECHNICAL FIELD

Methods pertain to treatment or mitigation of depression, particularlydepression-Parkinson's disease (PD) co-morbid condition. Particularly,methods comprise administering Carboxyl-terminal domain of the heavychain of tetanus toxin (Hc-TeTx) or any atoxic fraction of the tetanustoxin or the coding sequence of the Hc-TeTx in treatment or mitigationof depression.

BACKGROUND

The toll extracted by clinical depression, characterized by a despondentfeeling, loss of interest in pleasurable activities, guilt,worthlessness, and trouble concentrating, is of immense medical concern.This is because the prevalence is relatively high. In the U.S. alone,approximately 16 million people or 7% of the adults are afflicted withmajor depressive disorder, which may also include abnormalities inappetite and sleep and loss of productivity and suicidal ideation. Theactual suicide rate, estimated at 1 million worldwide, not only affectsthe afflicted individual but also the family and friends and at timesthe entire community (P. E. Greenberg, A. A. Fournier, T. Sisitsky, etal., The economic burden of adults with major depressive disorder in theUnited States, (2005 and 2010) J Clin Psychiatry, 76 (2015), pp.155-162).

Although our understanding of the highly complex neurobiologicalcircuitry of mood regulation remains far from complete, it is known thatthe symptoms of depression are diverse and vary from patient to patient.In addition, a number of drugs developed over the past six decades suchas, tricyclic antidepressants (TCAs), monoamine oxidase inhibitors(MAOIs), norepinephrine reuptake inhibitors (NRIs), and selectiveserotonin reuptake inhibitors (SSRIs) have offered significant relief toat least some of the patients (M. R. Levinstein, B. A. Samuels,Mechanisms underlying the antidepressant response and treatmentresistance, Front Behav Neurosci, 8 (2014), pp. 208). These medications,however, based on biogenic amine theory of depression, which posits thata decrease in these neurotransmitters is the primary cause of thedisorder, have several major drawbacks. These include: limited efficacy,delayed onset and various undesirable side effects, some of which may bepersistent (A. J. Rush, Targeting treatments for depression: what canour patients tell us? Epidemiol. Psychiatr Sci, 26 (2017), pp. 37-39; J.Ben-Sheetrit, D. Aizenberg, A. B. Csoka, et al., Post-SSRI sexualdysfunction: clinical characterization and preliminary assessment ofcontributory factors and dose-response relationship. J ClinPsychopharmacol, 35 (2015), pp. 273-278). Hence more rapid onsetantidepressants with wider efficacy and lower side effects are urgentlyneeded.

Recent elucidation of significant contribution of neurotrophic factorsand inflammatory processes in mood regulation/dysregulation, has pointednew approaches in development of more effective antidepressants. In thisregard, several natural and synthetic compounds with anti-inflammatoryproperties and ability to increase neurotrophic factors, particularlybrain-derived neurotrophic factor (BDNF) have been proposed as potentialnovel antidepressants (L. L. Hurley, Y. Tizabi, Neuroinflammation,neurodegeneration, and depression. Neurotox. Res, 23 (2013), pp.131-144; O. Kalejaiye, B. Getachew, C. L. Ferguson, et al.,Alcohol-Induced Increases in Inflammatory Cytokines Are Attenuated byNicotine in Region-Selective Manner in Male Rats. J Drug Alcohol Res,(2017), pp. 6: 236036; C. N. Bodnar, J. M. Morganti, A. D. Bachstetter,Depression following a traumatic brain injury: uncovering cytokinedysregulation as a pathogenic mechanism. Neural Regen Res., 13 (2018),pp. 1693-1704; R. S. Duman, BDNF, 5-HT, and anxiety: identification of acritical periadolescent developmental period. Am. J. Psychiatry, 174(2017), pp. 1137-1139). However, no study on the C-terminal domain ofthe heavy chain of tetanus toxin (Hc-TeTx) as a potential antidepressanthas been conducted.

SUMMARY OF THE INVENTION

The present inventors have conducted an extensive research and havediscovered that the C-terminal domain of the heavy chain of tetanustoxin (Hc-TeTx) has antidepressant effects in an animal model ofdepression and can be effective in treating depression, particularlydepression associated with Parkinson's disease (PD). The presentinventors have discovered that Hc-TeTx resulted in a dose-dependentdecrease in immobility score, whereas the open field locomotor activity(OFLA) was not affected. Concomitant with the behavioral effects, theinventors have discovered an increase in central brain-derivedneurotrophic factor (BDNF) but a decrease in tumor necrosis factor(TNF)-alpha (TNF-alpha) in the hippocampus and the frontal cortex, twoareas intimately associated with mood regulation (J. Jin and S. Maren,Prefrontal-hippocampal interactions in memory and emotion. FrontiersSystems Neuroscience, 9 (2015), pp. e170; Getachew, S. R. Hauser, A. B.Csoka et al., Role of cortical alpha-2 adrenoceptors in alcoholwithdrawal-induced depression and tricyclic antidepressants. DrugAlcohol Depend., 175 (2017), pp. 133-139). These results indicate longlasting antidepressant effects of Hc-TeTx and suggest potential utilityof Hc-TeTx in depression, particularly PD-depression co-morbidity.

Characteristics described above, other characteristics, and advantagesof the invention are clearly revealed with reference to the descriptionsbelow and accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows effects of various doses of Hc-TeTx on immobility in theforced swim test (1A) and open field locomotor activity (1B) in WKYrats. The animals were tested 24 h after the single i.m. injection.Values are mean±SEM. N=6/group. *p<0.05, **p<0.01 compared to control.

FIG. 2 shows effects of various doses of Hc-TeTx on immobility in theforced swim test in WKY rats. The animals were tested one week (2A) andtwo weeks (2B) after the single i.m. injection. Values are mean±SEM.N=6/group. *p<0.05, **p<0.01 compared to control.

FIG. 3 shows effects of Hc-TeTx on BDNF levels in the hippocampus(Hippo) and the frontal cortex (FCX) of WKY rats treated with 60 μg/kgHc-TeTx. The animals were sacrificed 24 h after the single i.m.injection of Hc-TeTx. Values are mean±SEM. N=6/group. **p<0.01 comparedto control.

FIG. 4 shows effects of Hc-TeTx on TNF-alpha levels in the hippocampus(Hippo) and the frontal cortex (FCX) of WKY rats treated with 60 μg/kgHc-TeTx. The animals were sacrificed 24 h after the single i.m.injection of Hc-TeTx. Values are mean±SEM. N=6/group. **p<0.01 comparedto control.

DETAILED DESCRIPTION OF THE INVENTION

Tetanus toxin is a synthesized single peptide of approximately 150 kDa,which consists of 1315 amino-acid residues. An endogen clostrialprotease forms a two-chain activated molecule composed of a heavy chain(HC) and a light chain (LC) linked by a disulfide bond. The catalyticdomain of the toxin resides in the LC, while the translocation andreceptor-binding domains are present in HC. The C-terminal domain ofheavy chain of tetanus toxin (Hc-TeTx) is a nontoxic fragment of TeTxwith demonstrated capacity to protect against cell death induced by avariety of neurotoxins including methamphetamine (I. Chaib-Oukadour, C.Gil, J. Rodriguez-Alvarez, et al., Tetanus toxin H(C) fragment reducesneuronal MPP+ toxicity. Mol Cell Neurosci, 41 (2009), pp. 297-303; L.Mendieta, B. Venegas, N. Moreno, et al., The carboxyl-terminal domain ofthe heavy chain of tetanus toxin prevents dopaminergic degeneration andimproves motor behavior in rats with striatal MPP(+)-lesions. NeurosciRes 65 (2009), pp. 98-106; A. Sanchez-Gonzalez, L. Mendieta, V. Palafox,et al., The restorative effect of intramuscular injection of tetanustoxin C-fragment in hemiparkinsonian rats. Neurosci Res, 84 (2014), pp.1-9. L; Mendieta, N. Granado, J. Aguilera, et al., Fragment C domain oftetanus toxin mitigates methamphetamine neurotoxicity and its motorconsequences in mice. Int J Neuropsychopharmacol, 19 (2016), pyw021; L.Radenovic, V. Selakovic, S. Olivan, et al., Neuroprotective efficiencyof tetanus toxin C fragment in model of global cerebral ischemia inMongolian gerbils. Brain Res Bull, 101 (2010), pp. 37-44; M. C.Sozbilen, M. Ozturk, G. Kaftan, et al., Neuroprotective effects ofC-terminal domain of tetanus toxin on rat brain against motorneurondamages after experimental spinal cord injury. Spine, (Phila Pa. 1976)43 (2018), pp. E327-E333). A strong association betweenneurodegenerative diseases (e.g. PD) and neuropsychiatric disorders(e.g. depression) in terms of neurobiological substrates as well as drugtreatment has been indicated (Y. Tizabi, Duality of Antidepressants andNeuroprotectants. Neurotox Res, 30 (2016), pp. 1-13).

The present inventors have undertaken, for the first time, the study toinvestigate the potential antidepressant effects of Hc-TeTx in an animalmodel of depression. The present inventors have discovered that Hc-TeTxresulted in a dose-dependent decrease in immobility score, whereas theopen field locomotor activity (OFLA) was not affected. Immobility in theforced swim test (FST) is a measure of helplessness or depressive-likebehavior (W. P. Pare, Open field, learned helplessness, conditioneddefensive burying, and forced-swim tests in WKY rats. Physiol Behav, 55(1994), pp. 433-439). Concomitant with the behavioral effects, theinventors have discovered an increase in central brain-derivedneurotrophic factor (BDNF) but a decrease in tumor necrosis factor(TNF)-alpha (TNF-alpha) in the hippocampus and the frontal cortex, twoareas intimately associated with mood. These results indicate longlasting antidepressant effects of Hc-TeTx and suggest potential utilityof Hc-TeTx in depression, particularly PD-depression co-morbidity.

One example embodiment of the disclosed subject matter provides an agentfor depression treatment or mitigation, containing C-terminal domain ofthe heavy chain of tetanus toxin (Hc-TeTx) as an active ingredient.

Another example embodiment of the disclosed subject matter provides amethod for treating or mitigating depression, comprising administeringan effective amount of Hc-TeTx to a patient in need thereof.

In one of its aspects, the depression is depression associated withParkinson's disease (PD) or PD-Depression co-morbidity.

In one of its aspects, Hc-TeTx increases central brain-derivedneurotrophic factor (BDNF) in the hippocampus and the frontal cortex.

In one of its aspects, Hc-TeTx decreases tumor necrosis factor(TNF)-alpha (TNF-alpha) in the hippocampus and the frontal cortex.

In one of its aspect, Hc-TeTx has long lasting effect, which would allowfor less frequent administration of the drug.

The formulation of Hc-TeTx and method of administration of Hc-TeTx canbe selected suitably. For example, the Hc-TeTx can be formulated in aliquid suitable for administration by injection or by nasal spray. Thisformulation can be produced by well-known methods.

The injection or nasal spray agents include solutions of solid agents tobe dissolved in a solvent before use. The injection or nasal spray agentis used by, for example, dissolving an active ingredient in a solvent.Examples of the solvent include distilled water for injection,physiological saline, etc. Furthermore, the injection or nasal sprayagent may contain a stabilizer, a dissolution aid, a suspending agent,an emulsifying agent, a soothing agent, a buffer, a preservative, andthe like. Such an injection agent is produced by sterilizing at thefinal step or employing an aseptic process.

The dose of the Hc-TeTx to be used is different depending on ages, bodyweights, symptoms, therapeutic effects, administration method, treatmenttime, and the like. For example, the dose of the Hc-TeTx per adult isgenerally from 150 μg to 600 μg per dose, in one aspect from 180 μg to540 μg per dose, and in another aspect from 180 μg to 360 μg per dose,once every two weeks by parenteral administration (most likelyintramuscular or it could be applied by a single intranasal dose).Needless to say, as mentioned above, the dose to be used variesdependent on various conditions. Therefore, the lowest dose specifiedabove may be sufficient in some cases, and a dose higher than the rangespecified above may be needed in some cases.

The Hc-TeTx may be administered in combination with other medicine (forexample, well-known agents for depression treatment) for the purposesof: (1) supplementing and/or enhancing therapeutic effect, (2) improvingthe kinetics, improving absorption, and reducing the dose; and/or (3)eliminating the adverse reaction of the compound.

EXAMPLES

The present invention is explained below in further detail withreference to Examples. However, the scope of the invention is notlimited to these Examples.

Wistar Kyoto (WKY) rats, an inbred strain, initially developed as anormotensive control for the spontaneously hypertensive rats, were laterfound to demonstrate exaggerated immobility in the forced swim test(FST), a measure of helplessness or depressive-like behavior (W. P.Pare, Open field, learned helplessness, conditioned defensive burying,and forced-swim tests in WKY rats. Physiol Behav, 55 (1994), pp.433-439). Moreover, it was found that these rats are irresponsive toselective serotonin reuptake inhibitors (SSRIs), and hence may beconsidered as a model of treatment resistant depression (C.Lopez-Rubalcava, I. Lucki, Strain-dependent modification of behaviorfollowing antidepressant treatment. Prog Neuropsychopharmacol BiolPsychiatry, 27 (2003), pp. 7-14; C. C. Will, E. E. Aird, F., Redei,selectively bred Wistar-Kyoto rats: an animal model of depression andhyper-responsiveness to antidepressants. Mol Psychiatry, 8 (2003), pp.925-932).

Adult male WKY rats, 14-15 weeks old and weighing about 250 g, wereobtained from Envigo (previously Harlan Laboratories, Indianapolis,Ind.). Animals receiving the same treatment were pair-housed through theduration of the experiment in a standard polypropylene shoebox cages(42×20.5×20 cm) on chip bedding. Animals were subjected to a 1-weekacclimatization period upon their arrival, during which they werehandled daily to minimize any handling related stress. Throughout thestudy, with the exception of behavioral tests, animals had free accessto food (Harlan Tek Lab) and water. The room was maintained at 24-26° C.at 55-66% relative humidity, on a reverse light cycle (lights on 7:00PM-7:00 AM) to allow convenient behavioral evaluations of the animalsduring their active period. Acclimatization to reversed dark cycle wasdone over a one-week period where the light hours were shifted byapproximately 2 h daily. All behavioral testing and injections occurredbetween 8:00 A.M. and 12:00 P.M. during the animal's active phase. Allexperiments were carried out in accordance with NIH guidelines, asapproved by the Institutional Animal Care and Use Committee of theHoward University.

Animals were divided into three groups (n=6/group) and receivedintramuscular (i.m.) injection of either saline (control) or 20, 40, or60 μg/kg dose of Hc-TeTx. The injection was into the gastrocnemiusmuscle. Hc-TeTx fragment was synthesized as described in detailpreviously (Herrando-Grabulosa M, Casas C, Aguilera J. The C-terminaldomain of tetanus toxin protects motoneurons against acute excitotoxicdamage on spinal cord organotypic cultures. J Neurochem. 2013;124(1):36-44) and indicated below.

Hc-TeTx Purification

Escherichia coli BL21 cells were transformed with pQE3 (Qiagen,Chatsworth, Calif., USA) vector encoding for (6×His)-tagged He-TeTx andwere grown in Luria Bertani medium containing 100 μg/mL ampicillin asreported previously (Gil C., Chaib-Oukadour I. and Aguilera J. (2003)C-terminal fragment of tetanus toxin heavy chain activates Akt andMEK/ERK signaling pathways in a Trk receptor-dependent manner incultured cortical neurons. Biocher. J. 373, 613-620). Protein expressionwas induced by the addition of 0.4 mM isopropyl β-D-thiogalactoside(IPTG). After 3 h, cells were pelleted by centrifugation at 4000 g for20 min at 4° C., re-suspended in lysis buffer (50 mM NaH₂PO₄, 300 mMNaCl, and 1% Triton-X-100; pH 8) and sonicated on ice for six 30 speriods. The suspension was centrifuged at 30000 g for 30 min at 4° C.The clear supernatant, which contains the His-tagged protein, waspurified by cobalt affinity chromatography. Mixed proteins were injectedin a Fast Protein Liquid Chromatography (FPLC), which contains acobalt-agarose resin (TALON Metal Affinity resin; Clontech Laboratories,Palo Alto, Calif., USA), previously equilibrated (50 mM NaH₂PO₄.H₂O and300 mM NaCl; pH 7). The proteins, without His-Tags, were eluted bywashing the resin with elution buffer (50 mM NaH₂PO₄.H₂O and 300 mMNaCl; pH 7). Hc-TeTx contains six histidines, which is retained in theresin forming a Co-complex. Hc-TeTx was eluted with the elution buffer(50 mM NaH₂PO₄.H₂O, 300 mM NaCl and 150 mM Imidazole; pH 7). Fractionscollected were 0.5 mL volume. The elution process can be followed withFPLC system, that measures the absorbance at 280 nm constantly. Proteinwas separated by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) at 12%. Gel was stained with GelCode BlueStain Reagent (Pierce Chemical Co., Rockford, Ill., USA) and thosefractions containing purified Hc-TeTx protein were dialyzed (40 mMNa₂HPO₄, 10 mM NaH₂PO₄ and 150 mM NaCl; pH 7.4), overnight at 4° C., andfor 2 h with new buffer. Protein concentrations were determined usingthe bicinchoninic acid assay (BCA; Pierce Chemical Co.) and lyophilized.The Hc-TeTx was stored in aliquots at −20° C.

In addition, to determine the absorption and uptake and subsequentarrival at the spinal cord level, Alexa Fluor®555 was attached toHc-TeTx following the manufacturer's protocol from Alexa Fluor®555Labeling Kit (Invitrogen, Carlsbad, Calif., USA) and stored at −20° C.

The Hc-TeTx solution was prepared by dissolving 1 mg of lyophilizedHc-TeTx in 1 mL of isotonic saline solution (0.9% HCl), followed byserial dilutions to obtain a final concentration of 20, 40 or 60 μg/100μL. The volume of injection was 400 L/kg. Hence each animal receivedapproximately 100 μL of saline or the drug.

Approximately 24 h after the last injection, animals were tested in anopen-field activity monitoring cage (27×27×20.3 cm, Med Associates,Inc., St. Albans, Vt.) for 5 min where ambulatory counts representingthe number of infrared beam interruptions were recorded. This behaviorwas assessed to determine if drug treatment affected general locomotorbehavior, which might impinge on forced swim test immobility assessment(L. Akinfiresoye, Y. Tizabi, Antidepressant effects of AMPA and ketaminecombination: role of hippocampal BDNF, synapsin, and mTOR.Psychopharmacology (Berl), 230 (2013), pp. 291-298; B. Getachew, Y.Tizabi, Both ketamine and NBQX attenuate alcohol-withdrawal induceddepression in male rats. J Drug Alc Res, 8 (2019), pp. 1-7).

Immediately following the open field activity test each animal wasevaluated for its behavior (immobility) in Forced Swimming Test (FST).Briefly, each rat was placed in a Pyrex cylinder pool measuring 17 cm indiameter and 60 cm in height for 5 min. The cylinder was filled with 30cm water (25±1° C.) to ensure that the animals could not touch thebottom of the container with their hind paws or their tails. The FSTactivity was video recorded for subsequent analysis. The rat was removedafter 5 min, dried, and placed in its home cage. A time sampling scoringtechnique was used whereby the predominant behavior in each 5-s periodof the 300-s test was recorded. Inactivity (immobility) and activity(swimming) were distinguished as mutually exclusive behavioral states.Swimming behavior was defined as movement (usually horizontal)throughout the cylinder. Immobility was defined when no additionalactivity was observed other than that required to keep the rat's headabove the water.

Since behavioral effects were observed a day after a single Hc-TeTxinjection, both OFLA and FST were repeated after one week of rest andagain after two weeks of rest to determine the lasting effects of thesingle drug injection on these parameters.

A separate group of rats were treated with the 60 μg/kg dose of Hc-TeTxas this dose had resulted in the highest behavioral (antidepressant)effect. Animals were sacrificed by decapitation, approximately 24 hlater to coincide with the time of behavioral observation. No behavioraltests were done in these animals. This was to avoid potentialconfounding effects of swim test on neurochemical parameters. Brainswere quickly removed, frozen on dry ice and stored at −80° C. untildissection for BDNF and TNF-alpha measurement. The hippocampus(bilateral) and frontal cortex were dissected as described (Tizabi Y,Getachew B, Rezvani A H, Hauser S R, Overstreet D H. Antidepressant-likeeffects of nicotine and reduced nicotinic receptor binding in theFawn-Hooded rat, an animal model of co-morbid depression and alcoholism.Prog Neuropsychopharmacol Biol Psychiatry. 2009; 33(3):398-402) anddetailed below. Brains were thawed and kept on an ice-cold plate.Frontal cortex (up to the genu of corpus callosum and excluding theolfactory bulb and olfactory tubercle), and hippocampus (bilateral) wereremoved and stored frozen at −80° C. until assayed.

Western blot was performed as described in detail in the following twodocuments, the disclosures of which are incorporated herein byreference: B. Getachew, S. R. Hauser, A. B. Csoka et al., Role ofcortical alpha-2 adrenoceptors in alcohol withdrawal-induced depressionand tricyclic antidepressants. Drug Alcohol Depend., 175 (2017), pp.133-139; and L. Akinfiresoye, Y. Tizabi, Antidepressant effects of AMPAand ketamine combination: role of hippocampal BDNF, synapsin, and mTOR.Psychopharmacology (Berl), 230 (2013), pp. 291-298). Briefly, homogenateof the dissected hippocampus (bilateral) were made in lysis buffer (10mM Tris-buffer, 5 mM EDTA, 150 mM NaCl, 0.5% Triton X-100 (v/v) withprotease inhibitors (Sigma-Aldrich, St. Louis, Mo.). The proteinconcentration in each sample was determined using a BCA protein AssayKit (Pierce Biotechnology Inc., IL), and equal protein amount (asconfirmed by β-actin) was loaded in each immunoblot. The proteins wereseparated using 12% SDS-PAGE gel and transferred onto a nitrocellulosemembrane. The membranes were blocked with a blocking reagent (5% nonfatmilk in TBS buffer) for ½ h and incubated at 4° C. overnight with theprimary antibody against BDNF (1:500, Santa Cruz Biotechnology Inc.,Santa Cruz, Calif.) or TNF-alpha (1:500, Santa Cruz Biotechnology). Themembranes were washed with TBST (TBS buffer with 1% Tween-20) andblocked with the blocking reagent. Membranes were then incubated for 1 hat room temperature in Goat Anti-Rabbit-HRP conjugated secondaryantibody (1:3000 in TBS, Bio-Rad Laboratories, CA). The membranes werethen washed in the TBST washing solution and then visualized usingenhanced chemiluminescent kits (Bio-Rad Laboratories, CA). The intensityof the protein bands on the gel was quantified using ChemiDoc XRS system(Bio-Rad Laboratories, CA).

Statistical differences between treatment groups were determined byone-way analysis of variance (ANOVA) followed by post-hoc Newman-KeulsMultiple comparison test to determine which groups differed. Significantdifference was set a priori at p<0.05. Data were analyzed using GraphpadPrism6 (Graphpad Software, Inc., San Diego, Calif., USA).

Single treatment with Hc-TeTx resulted in a dose-dependent decrease inFST immobility when tested 24 h after the injection [F(3,28)=6.38,p<0.01]. Thus, the 60 μg/kg dose caused the highest decrease (60%p<0.01), the 40 μg/kg (51% p<0.01) and the 20 μg/kg (21% p<0.05) inimmobility compared to the control (FIG. 1A). Open field locomotoractivity was not altered by any treatment (FIG. 1B), suggesting that thetreatment effects of Hc-TeTx on FST were independent of any effects ongeneral locomotion.

Based on these results, the present inventors used the highest dose of60 μg/kg to evaluate the neurochemical changes associated with thisbehavioral effect.

One week after the last single injection, the effect of 40 and 60 μg/kgdoses were still evident on immobility scores [F(3,28)=5.96, p<0.01].Hence, with the 40 μg/kg dose, there was 29% decrease in immobility(p<0.05) and with 60 μg/kg dose, there was 39% decrease (p<0.01) (FIG.2A). After 2 weeks of rest the effect of 40 μg/kg dose was totallyabsent (FIG. 2B). Although there was still a 13% decrease in immobilityscore after the 60 μg/kg dose, this effect was not statisticallysignificant [F(3,28)=0.68, p>0.64].

Western blot analysis showed that acute treatment with the 60 μg/kg doseof Hc-TeTx resulted in increases in BDNF levels in the hippocampal(2.6-fold, p<0.01) and the frontal cortex (2.1-fold p<0.01) 24 h after asingle administration (FIG. 3). An opposite trend was observed in termsof TNF-alpha levels in both areas. Hence, 60 μg/kg dose of Hc-TeTxresulted in decreases in TNF-alpha levels in the hippocampal (2.5-fold,p<0.01) and the frontal cortex (5-fold p<0.01) 24 h after a singleadministration (FIG. 4).

The results suggest antidepressant-like effects of an acute dose ofHc-TeTx in an animal model of treatment-resistant depression. Thiseffect was long lasting as the behavioral despair reflected in theimmobility scores of the FST was still down one week after theinjection. Since potential utility of Hc-TeTx in movement disordersassociated with PD has been verified by a number of preclinical studied(F. Patricio, I. Parra, I. Martinez, et al., Effectiveness of fragment Cdomain of tetanus toxin and pramipexole in an animal model ofParkinson's disease. Neurotox Res, 35 (2019), pp. 699-710; L. Mendieta,E. Bautista, A. Sanchez, et al., The C-terminal domain of the heavychain of tetanus toxin given by intramuscular injection causesneuroprotection and improves the motor behavior in rats treated with6-hydroxydopamine. Neurosci Res, 74 (2012), pp. 156-167) andco-morbidity of depression with PD is also well established (R. M. J.van der Velden, M. P. G. Broen, M. L. Kuijf, et al., Frequency of moodand anxiety fluctuations in Parkinson's disease patients with motorfluctuations: A systematic review. Mov Disord, 33 (2018), pp.1521-1527), it may be concluded that Hc-TeTx would be of specificbenefit in such co-morbid condition. This contention is furthersupported by the findings that neuroprotectants in general, are likelyto have antidepressant effects as well (Y. Tizabi, Duality ofAntidepressants and Neuroprotectants. Neurotox Res, 30 (2016), pp.1-13).

The results also implicate a role for the neurotrophic factor, BDNF andat least one of the pro-inflammatory cytokines, TNF-alpha inantidepressant effects of Hc-TeTx. This is due to the fact the levels ofthe BDNF in both hippocampus and the frontal cortex were elevated byHc-TeTx, whereas the levels of TNF-alpha were reduced in both theseareas a day after the drug injection, concomitant with the observedantidepressant effects.

In summary, adult male Wistar-Kyoto rats, a putative animal model ofdepression, were treated with various doses of Hc-TeTx (0, 20, 40 and 60μg/kg, IM) and their performance in the open field locomotor activity(OFLA) as well as in the forced swim test (FST) was evaluated at 24 h,one week and two weeks after the single injection. A separate group ofrats were injected with 60 μg/kg Hc-TeTx and sacrificed 24 h later forneurochemical evaluations. Hc-TeTx resulted in a dose-dependent decreasein immobility score after 24 h, whereas OFLA was not affected.Concomitant with the 24 h behavioral effects, the levels of hippocampaland frontal cortical BDNF were significantly increased, whereas thelevels of TNF-alpha in both these areas were significantly decreased.The decrease in immobility scores following higher doses of Hc-TeTx werestill evident after one week, but not 2 weeks of rest. These resultsindicate long lasting antidepressant effects of a single Hc-TeTx doseand suggest potential utility of Hc-TeTx as a novel intervention inPD-depression co-morbid condition.

Although current approved antidepressants are primarily based on themonoaminergic hypothesis, which posits that a decrease in the levels ofneurotransmitters such as norepinephrine, dopamine and serotonin (5HT)in the brain, is responsible for mood dysregulation, the delay in onsetof action of such antidepressants and their limited efficacy has shiftedthe focus to other potential biological substrates. In this regard, arole for neurotrophic factors, particularly hippocampal and also frontalcortical BDNF and more recently, dysregulation of immune system,reflected in elevated levels of pro-inflammatory cytokines such asTNF-alpha have gained substantial traction in the field. Thus, it is nowhypothesized that the delay in onset of action of currentantidepressants might be due to the delay in elevation of theneurotrophic factors. Moreover, inhibition of both basal and stimulatedserotonin uptakes in primary neuronal cultures were demonstrated byHc-TeTx. Also, in-vivo studies show that Hc-TeTx increases the activityof tryptophan hydroxylase, a key enzyme in the synthesis of serotonin,which can lead to maintaining high levels of serotonin in the centralnervous system.

While the subject matter disclosed herein has been described inconnection with what is presently considered to be practical exampleembodiments, it is to be understood that the present disclosure is notlimited to the disclosed embodiments, and covers various modificationsand equivalent arrangements included within the spirit and scope of theappended claims.

What is claimed is:
 1. A method for treating or mitigating depression,comprising administrating an effective amount of C-terminal domain ofthe heavy chain of tetanus toxin (Hc-TeTx) to a subject in need thereof.2. The method according to claim 1, wherein the depression is depressionassociated with Parkinson's disease (PD).
 3. The method according toclaim 1, wherein the Hc-TeTx is administered by injection.
 4. The methodaccording to claim 1, wherein the Hc-TeTx is administered in an amountof 150 μg to 600 μg per dose.
 5. The method according to claim 1,wherein the Hc-TeTx is administered in an amount of 180 μg to 540 μg perdose.